Molecular Fingerprinting and Phytochemical Investigation of Syzygium cumini L. from Different Agro-Ecological Zones of India

Syzygium cumini L. (ver Jamun; BlackBerry) is a native, evergreen multipurpose tree species of India. Besides being a fruit tree and for agroforestry in different regions, it is medicinally important too. This study aimed to determine genetic diversity using molecular and phytochemical markers in sixteen genotypes of Indian S. cumini from different agro-ecological zones. The present study used a combination of ISSR markers and the HPLC technique to explore these genotypes. The results showed a wide genetic diversity range based on the similarity coefficient values observed in S. cumini sixteen accessions from different sites. Four primary phenolic acids were discovered in all the accessions; caffeic acid (CA) was found in high concentrations. The intraspecific association between molecular and phytochemical characteristics was the primary goal of this investigation. By employing gene-specific markers for the route of secondary metabolites (polyphenols) production, it further investigated the progressive research of diversity analysis of polyphenol content in S. cumini accessions, which may also expand its nutraceutical and pharmaceutical utilization.


Introduction
Biodiversity loss is now one of the world's most pressing crises, and parts of the world endowed with rich genetic resources are facing grim scenarios with the alarming rate of loss of biodiversity. IUCN recognizes inadequate coverage of evaluations across all plant categories to report an accurate percentage of vulnerable species for plants. In total, 54% of plant species are currently recognized on the IUCN Red List as threatened [1]. Further, more than half of the vascular plant species in the world are currently found in 35 hotspots [2]. Ecosystem diversity, species diversity, and genetic diversity are the three categories used to categorize biological variety, and efforts are being made at all levels to conserve biodiversity. In addition to being essential for ecological and evolutionary research, the sum and circulation of genetic diversity within and among populations also form the basis for functional genomic investigations [3].
Syzygium cumini L. (ver Jamun; BlackBerry), a native, evergreen tree species of India, thrives simply in a tropical climate and is found not only in numerous parts of the Indian sub-continent but also several other countries of Asia and Eastern Africa [4]. In India, the tree is widely grown in the Indo-Gangetic plains and the Cauvery delta of Tamil Nadu [5]. Besides being important as a fruit tree and for agroforestry in different regions, S. cumini is medicinally important too, and over many decades, traditional practitioners have employed various methods to manage diabetes [6]. Despite having a high economic worth, nothing is  Genomic DNA was extracted using the CTAB procedure with certain modifications [13,14]. The amount of DNA isolated was measured using agarose gel electrophoresis. Twentysix ISSR markers (Table 2) were employed in the molecular marker analysis to characterize 16 distinct accessions of S. cumini plant samples. ISSR analysis was performed using genomic DNA at a concentration of 25 ng. DNA amplifications were performed using a thermocycler (PQ lab-Primus 96) for the ISSR analysis. One negative control (master mix with water in place of DNA template) was added to check for contamination in the experiment. All the molecular analyses were conducted in three replicates. A submerged gel electrophoresis midi unit from Bangalore Genei Pvt. Ltd. was used for fractionating the PCR products on an agarose gel. A low-range DNA ruler was included on one side of the gel as a molecular standard. The gel was visualized on a UV transilluminator (Bio-Rad, USA), photographed, and analyzed using the Kodak gel documentation system (Model EDAS 290) using Kodak ID Image analysis software.

Phytochemical Study of S. cumini
This study generated a chem profile of different S. cumini germplasm with chromatographic techniques TLC and HPLC.

1.
Extraction and estimation of polyphenols The Folin-Ciocalteu technique was expended to ascertain the total polyphenol contented (TPC) [15]. Samples of leaves were made at a concentration of 1 g in 10 mL of buffer. The incubation of the mixture was done for 90 min at 25 • C and analyzed by UV-Vis spectrophotometer at 725 nm.

2.
Separation of polyphenols using TLC For the TLC analysis, standards (caffeic acid, ferulic acid, o-coumaric acids, sinapic acid, and p-coumaric acids) and polyphenol samples were exposed to qualitative TLC analysis on cellulose plates (20 × 20 cm, Merck, Darmstadt, Germany). Extracted polyphenol samples (20 µL) were spotted on the plate and developed under the following mobile phase conditions: (1)

3.
Separation of polyphenols using HPLC Standards (caffeic acid, ferulic acid, o-coumaric acids, sinapic acid, and p-coumaric acids) stock solutions of 1 mg/mL concentration were arranged. The samples were examined using an HPLC system (Shimadzu, LC10A, Kyoto, Japan) with solvents A (2% aqueous glacial acetic acid) and B (30% acetonitrile and 2% aqueous glacial acetic acid), along with an analog SCL 10 AVP pump, an injection (20 µL loop), and an SPD 10 AVP detector. Data acquisition was made using Class VP Software. Chromatography was performed on a C8 column (250 × 4.6 mm; particle size 5 µm; Phenomenex, Torrance, CA, USA). A maximum pressure of 400 kgf/cm 2 and a minimum of 0 Kgf/cm 2 were sustained, with a solvent ratio of 30:70 using A and B solvents, respectively, at a wavelength of 320 nm and 1 mL of flow rate with a binary mode of gradient system. Peak areas and retention times were computed using a Pentium computer and a Shimadzu Class-VP integrator.

Statistical Analysis
The ISSR data cast off to accumulate a separate binary matrix for cluster examination using the NTSYS-pc 2.1 [16]. The SIMQUAL (Similarity for Qualitative Data) procedure assessed genetic similarity between collections according to Jaccard's similarity coefficient [17]. The dendrogram was then created using the similarity coefficients and the UPGMA (Unweighted Pair Group Method with Arithmetical Averages) function of the NTSYS-pc 2.1 package's SAHN (Sequential Agglomerative Hierarchical and Nested Clustering) routine. The dendrogram was graphically signified as a phenetic tree through the TREE program NTSYS-pc 2.1 package. Finally, the binary data based on Nie's coefficient matrix was used to calculate principal component analysis (PCA) with the same program using the EIGEN and PROJ modules to highlight the resolving power of ordination. The percentage of polymorphic bands (PPB) and resolving power (Rp) were calculated [18]. Rp and polymorphism information content (PIC) or gene diversity value were calculated using the method [19]. According to the indices, the information content of each marker system was established [20]. Conclusively, the gathered data from the molecular and phytochemical analysis of S. cumini samples the correlation analysis was performed using XLSTAT-2015 software [21].

DNA Extraction Analysis
Though the plant species are the same, the molecular weight of genomic DNA ranged from 19.5 kb to 20.4 kb ( Table 3). Kulkarni et al. [22] reported that isolation of genomic DNA from S. cumini was challenging because of high levels of polyphenols, tannins, and polysaccharides, which on cell disruption, form a sticky gelation in which nucleic acids become embedded. However, in the present case, overall, a good yield of genomic DNA was obtained from most of the samples of S. cumini collected from different locations. Genetic DNA, yield, and purity variations could be explained as each plant is grown in   (Table 4). A high percentage of polymorphic fragments (96.93%) were found in the PCR amplification using ISSR markers. It was predicted that the ISSR approach boosts possibly polymorphic microsatellite regions [23]. The number of bands speckled from 7 (22BV17 T8) to 17 (17BV18 T3), with a mean of 12.25 bands per primer. The size of the amplification product varied from 200-2500 bp. The resolving power of ISSR primers varied from 6.25 (20BV17A6) to 14 (15BV17C1), with an average resolving power of 10.08 (Table 4). The resolving power of primer 15BV17C1 was found to be 14; hence, it could be considered the most informative in terms of resolving power and the capacity of primers to distinguish between all accessions. Thus, it showed that the special price of resolving power designated the capability of primers to resolve the diverse, thoroughly linked accessions of S. cumini. Our consequences suggested that dinucleotide and trinucleotide ISSR occur at high frequencies.

ISSR Marker-Based Genetic Similarity and Cluster Analysis
Correspondence indices differentiated all the S. cumini accessions and directed a fair variety of unpredictability (0.45 to 0.90) in the resemblance coefficient values, signifying a broad genetic base of 16 genotypes investigated in the present study. Accessions from Saharanpur (CD-5.4) and Meerut (D-4.3) that belonged to dry sub-humid and semi-arid zone, respectively, were found most similar to each other (similarity coefficient 0.89). Similarly, samples collected from neighboring cities of Meerut (D-4.3) and Kanpur (CD-5.4), which too belonged to dry sub-humid and semi-arid zone, respectively, had a high similarity coefficient (0.87). The last value of similarity coefficient (0.45) was detected between the genotypes from Nazibabad (CD-6.1) and Kota (D-4.2), which fitted to moist sub-humid and semi-arid zone, respectively. Similarly, Kota (D-4.2) and Bangalore (D-4.4) belonged to the same zone, i.e., semi-arid had a low similarity coefficient value (0.47) ( Table 4). Among sixteen samples of S. cumini, genetic similarity values were distributed in an acceptable range and displayed maxima at 0.5 similarity coefficient values. The current study proposes that ISSR is suitable for genetic diversity in tree species.
The UPGMA method was used to build a dendrogram based on the resemblance matrix expressing Jaccard's coefficient to assess the relationship between the genotypes. The dendrogram ( Figure 2) shows that 16 genotypes from various places created four clusters. However, not even one of the discrete primers could group these plants into agroclimatic sub-zone or region-specific clusters. Cluster one was represented by Jodhpur (E-1. Despite the substantial diversity among the samples, UPGMA analysis did not reveal grouping built on areas or agro-climatic sub-zones. Apart from cluster analysis, the principal component analysis (PCA) was performed on group accessions (Figure 3). Clustering was useful in detecting relationships among ecotypes, while PCA allowed a view of the relationships between groups. The genetic difference did not exhibit any geographical pattern, and there was no discernible relationship between genetic and geographic distance, according to Despite the substantial diversity among the samples, UPGMA analysis did not reveal grouping built on areas or agro-climatic sub-zones. Apart from cluster analysis, the principal component analysis (PCA) was performed on group accessions ( Figure 3). Clustering was useful in detecting relationships among ecotypes, while PCA allowed a view of the relationships between groups. The genetic difference did not exhibit any geographical pattern, and there was no discernible relationship between genetic and geographic distance, according to the PCA and UPGMA dendrogram. Despite the substantial diversity among the samples, UPGMA analysis did not reveal grouping built on areas or agro-climatic sub-zones. Apart from cluster analysis, the principal component analysis (PCA) was performed on group accessions (Figure 3). Clustering was useful in detecting relationships among ecotypes, while PCA allowed a view of the relationships between groups. The genetic difference did not exhibit any geographical pattern, and there was no discernible relationship between genetic and geographic distance, according to the PCA and UPGMA dendrogram.

Shannon Diversity Index Based upon ISSR Markers
Based upon ISSR variation, Table 5 presents the Shannon phenotypic diversity for S. cumini populations. The Hο within the semi-arid region ranged from 1.17 to 4.09, with a mean value of 2.65. Similarly, the lower and higher values of Hο for dry sub-humid and moist sub-humid varied from 0.69 to 2.16 and 1.18 to 2.56, respectively. The means of Hο for these two regions are 1.82 and 2.04, correspondingly. The Hο might not be analyzed for the arid zone, as only one sample was from this district. The average diversity at a population level (Hcol) varied from a lower value of 1.03 to a higher value of 1.86 with a mean value of 1.54, whereas the observed genetic diversity at the species level (Hsp) ranged from 2.48 to 3.98 with a mean value of 3.00. A comparable value of diversity at the

Shannon Diversity Index Based upon ISSR Markers
Based upon ISSR variation, Table 5  might not be analyzed for the arid zone, as only one sample was from this district. The average diversity at a population level (Hcol) varied from a lower value of 1.03 to a higher value of 1.86 with a mean value of 1.54, whereas the observed genetic diversity at the species level (Hsp) ranged from 2.48 to 3.98 with a mean value of 3.00. A comparable value of diversity at the species level (4.1 to 7.9 with a mean value of 6.07) is reported in Mulberry [24]. The coefficient of genetic differentiation (Gst) varied from a lower value of 0.38 to a higher value of 0.58 with an average of 0.46, which specified a high degree of genetic differentiation among populations. It presented that 46% of genetic variation exists among populations and 54% within the inhabitants. For woody plants, wind and cross-pollination accounted for 10% of genetic diversity among communities [25]. The estimates were greater than the average for several reasons, including the longer geographic distance between the four groups and natural environmental selection.
Nm, or the degree of gene flow, was only calculated to be 0.30. The result is comparable with earlier studies, as the reported value of gene flow was 0.44 in Changium smyrmiodes [26] and 0.24 in Eurya nitida [27]. In contrast, relatively higher values were reported as 0.6 in Phaseolus [28] and 0.91 in Mikania [29]. The little gene flow between populations may cause significant genetic divergence in this species. A lower level of gene flow is further supported by the high partitioning of heterozygosity in groups and could be exploited at intra and inter-group levels. Geographical environments, ecological considerations, and the plant's mating system likely shaped the increased genetic diversity within groups.
The estimates of Nm were 1.0 for S. cumini populations, indicating sufficient gene flow across populations to offset the effects of random drift. Genetic drift affects the genetic structure and heightens population divergence when populations are small and separate [30]. Consequently, reports of genetic divergence between populations have been made [31][32][33].

Total Phenol
Each plant's leaf methanolic extract's absorbance was measured at 725 nm, and the number of entire phenolics was calculated in terms of OD units. gdw −1 . The Folin-Ciocalteu colorimetric assay in this study detects all hydroxylated phenolic compounds. The total phenolics in diverse samples of S. cumini are represented in Table 6; Figure 4. The highest level of total polyphenols was recorded in the Varanasi sample (15.11 OD units.gdw −1 ), followed by Jodhpur (13.16 OD units.gdw −1 ) sample. It is understandable because these samples belong to dry sub-humid and arid zones, with water stress conditions and stress plants producing higher polyphenol content [34]. species, Hcol/Hsp = percentage of diversity within collections, Gst = [Hsp-Hcol]/Hsp = Diversity between collections, Nm = Gene flow.
Nm, or the degree of gene flow, was only calculated to be 0.30. The result is comparable with earlier studies, as the reported value of gene flow was 0.44 in Changium smyrmiodes [26] and 0.24 in Eurya nitida [27]. In contrast, relatively higher values were reported as 0.6 in Phaseolus [28] and 0.91 in Mikania [29]. The little gene flow between populations may cause significant genetic divergence in this species. A lower level of gene flow is further supported by the high partitioning of heterozygosity in groups and could be exploited at intra and inter-group levels. Geographical environments, ecological considerations, and the plant's mating system likely shaped the increased genetic diversity within groups.
The estimates of Nm were 1.0 for S. cumini populations, indicating sufficient gene flow across populations to offset the effects of random drift. Genetic drift affects the genetic structure and heightens population divergence when populations are small and separate [30]. Consequently, reports of genetic divergence between populations have been made [31][32][33].

Total Phenol
Each plant's leaf methanolic extract's absorbance was measured at 725 nm, and the number of entire phenolics was calculated in terms of OD units. gdw −1 . The Folin-Ciocalteu colorimetric assay in this study detects all hydroxylated phenolic compounds. The total phenolics in diverse samples of S. cumini are represented in Table 6; Figure 4. The highest level of total polyphenols was recorded in the Varanasi sample (15.11 OD units.gdw −1 ), followed by Jodhpur (13.16 OD units.gdw −1 ) sample. It is understandable because these samples belong to dry sub-humid and arid zones, with water stress conditions and stress plants producing higher polyphenol content [34].  The total phenolics in the samples that belonged to semi-arid regions were 8.77, 7.38, 9.04, 7.65, 10.57, 11.74, 12.75, 11.25 OD units.gdw −1 in Bhilwara, Kota, Jhalawar, Pratapgarh, Bhopal, Meerut, Banasthali, and Bangalore respectively. The total phenolics in the samples from the dry sub-humid region were 10.01, 11.12, 9.96, and 15.11 OD units.gdw −1 in Saharanpur, Kanpur, Lucknow, and Varanasi, respectively. In the moist sub-humid region samples, the phenolic content was higher, i.e., 9.36, 10.62, and 11.77 OD units.gdw −1 in Pantnagar, Nazibabad, and Roorkee, respectively. The polyphenol levels were particularly low in Pratapgarh, where total phenolics were only 7.65 OD units.gdw −1 . Thus, substantial differences in total phenolics between diverse accessions of S. cumini were observed, although the variations within various regions were relatively low. The dry sub-humid region showed a relatively broader range of 9.96-15.11 OD units.gdw −1 in total phenolics. The distribution of total phenolics among 16 S. cumini accessions revealed that the arid zone had the highest concentration of phenolics, followed by moist sub-humid, semi-arid, and dry sub-humid regions. The varying amount of total phenolics from the leaves of S. cumini may be a function of the vegetational stage, the geographic position of plants, and climatic circumstances [35,36].

Qualitative Analysis of Total Phenols Using TLC
Out of various tested solvent systems, 1, 5, 8, and 9 separated the standards of five phenolic acids, viz. ferulic acid, caffeic acid, sinapic acid, o-coumaric acid, and p-coumaric acid. The typical phenolic acid combination was divided into solvent system-1, which included benzene, acetic acid, and water, out of all the examined solvent systems (37:45:18). Table 7 lists the Rf values for several phenolic acid standards in various solvent arrangements. Based on the better resolution, the samples were permissible to run in solvent systems 1 and 8. After treatment with FeCl 3 , a blackish-green and yellow spot was distinctly observed in S. cumini. P-coumaric acid and caffeic acid were separated in solvent 8. Consequently, in the qualitative analysis of total phenolics using preparative TLC, only caffeic acid and p-coumaric acid were tentatively recognized in around accessions of S. cumini samples.

Qualitative Analysis of Phenolic Acids Using HPLC
The HPLC analysis showed variations in the qualitative and quantitative distribution of tentatively identified p-coumaric acid, caffeic acid, ferulic acid, and other unidentified compounds in different accession of S. cumini. The HPLC chromatograms of the five phenolic acids (ferulic acid, caffeic acid, p-coumaric acid, o-coumaric acid, and sinapic acid) and standard mixtures were monitored at 320 nm (Supplementary Figure S3). The retention time (RT) and areas of peaks for the standard phenolic acids used are given in Table 8. The RT of caffeic acid, ferulic acid, sinapic acid, o-coumaric acid, and p-coumaric acid were 6.88, 9.4, 9.07, 9.09, and 12.32, respectively. Comparing RT with available standards, the compounds as peak numbers 3, 4, and 8 (RT 9.4, 9.1, and 6.8) were tentatively identified as ferulic acid, p-coumaric, and caffeic acid, respectively. For further confirmation, the sample from Varanasi and Jhalawar was cochromatographed with caffeic acid and ferulic acid, respectively. The co-chromatography with authentic phenolic acid showed that the chromatograms' peaks were separated with a simultaneous increase in the integration area value. Moreover, a significant amount of other phenolic compounds was also present, but these could not be further identified.
The concentration of each compound (identified/unidentified) was calculated based on peak area. Detailed quantification of diverse phenolic compounds (mg.gdwt −1 ) is concise in Table 9. The caffeic acid, which varied in its amount from 0.56 mg.gdwt −1 in Bhilwara to 7.14 mg.gdwt −1 in Lucknow, was present in all samples. The p-coumaric acid varied from 0.02 mg.gdwt −1 in Bhilwara to 0.26 mg.gdwt −1 in Jodhpur and was also present in all samples of S. cumini. On the contrary, tentatively identified ferulic acid was present only in some samples and ranged from a lower amount of 0.04 mg.gdwt −1 in Jodhpur to a higher amount of 2.31 mg.gdwt −1 in Roorkee. The ferulic acid yield was exceptionally high in Roorkee. Based on their quantity, peaks 3 and 4 (RT 4.4 and 5.5) gave major and clear peaks of polyphenols with a relatively much higher percentage of area. The amount of unidentified compound as peak number 3 varied from 0.54 mg.gdwt −1 in Bhilwara (semi-arid) to 6.63 mg.gdwt −1 in Nazibabad (moist sub-humid). The peak 4 of the unknown compound originated at RT 5.5 and was enormous and ranged from 0.58 mg.gdwt −1 in Bhilwara (semi-arid) to 6.26 mg.gdwt −1 in Nazibabad (moist sub-humid). These two compounds were present in all accessions of S. cumini. Moreover, the other unidentified compounds as peak numbers 1, 2, 5, 6, 7, 9, 10, 11, 12 (RT 3.6, 3.9, 6.1, 6.3, 6.5, 7.3, 8.1, 8.4, and 8.6) were also observed in relatively lower but significant amount and were not presented in all samples.
Phenolic acids commonly found in wild medicinal plants are mainly caffeic acid, ferulic acid, p-coumaric, and sinapic acid. A comparable amount of caffeic acid and p-coumaric acid, 0.22 mg.g −1 and 0.77 mg.g −1, respectively, was reported in Eucalyptus uropendis [37]. A study showed the concentration of caffeic acid (2.4 to 11.4 mg/L) and p-coumaric acid (0.0 and 3.0 mg/L) in different cultivars of pear juice [38]. On the contrary, lower amounts of caffeic acid (0-0.4-mg.g −1 ) were reported in aromatic herbs [39]. Similarly, high values of p-coumaric acid 0.6 (mg/L) and caffeic acid 5.1 (mg/L) were also reported in apple juices [40]. Table 10 shows the concentration of caffeic acid, ferulic acid, and p-coumaric acid in different accessions of S. cumini.
A helpful technique for categorization of the "polyphenolic fingerprint" could be used [41]. It is characteristic of most plants because each species has a unique phenolic composition frequently used in chemotaxonomy [42,43]. There is no significant distribution pattern observed. However, specific chemotypes within the studied population of S. cumini from different geographic areas may be used in the manufacturing of phytomedicine for predicting phytochemical content in cultivar development. Moreover, it would also be helpful for plant breeding, quality control, and intellectual property rights (IPR).

Correlation between Phytochemical and Molecular Profiling
The focus of this study was to check the correlation of molecular and phytochemical characters at the intraspecific level. Phytochemical profiling was employed as the base marker for chemotypic clustering. The dendrogram based on chemical profiling is shown in Figure 5. The dendrogram based on chromatogram grouped the samples into two major clusters with two samples as independent branches. As can be seen from the dendrogram, the grouping of samples did not follow any pattern related to their agro-ecological region or geographical distance. The dendrogram based on chromatogram was compared with that generated using the molecular technique ISSR and showed little correlation. Samples from Pratapgarh and Banasthali, Chittorgarh, and Roorkee showed similarity in chemotyping and ISSR. Despite some similarities, a distinct correlation between the grouping of samples based on chemoprofiling and molecular profiling could not be established.
A study conducted in 2005 also could not correlate the genetic dendrogram with that based on essential oil content in Cymbopogon sp. [44]. This is understandable as chemoprofiling is influenced by the related climatic condition, geographic location, and vegetational stage of plants and leaves [44], whereas molecular profiling completely depicts the genetic structure of the plant. Conversely, a close relation was also reported in chemical and genetic variation of the Vitex rotundifolia population from different locations in China [45]. In 2006, another study reported a strong correlation between secondary metabolite content and the genetic characterization of six Hypericum species from Serbia [46].
The presented data imply that the morphotypes are of multiple origins or due to different ecological growing conditions rather than genetically determined and that phytochemical races are induced by a limited number of genetic differences, which might have occurred independently in different S. cumini populations. Analysis of genetic profiles, the type and content of polyphenols produced in each of the accessions of S. cumini could constitute valuable tools for analyzing this specie's biodiversity. Since these analyses successfully discriminate between the accessions of this species, they constitute interesting tools to select those accessions with the potential to be used for specific crop improvement programs. We explored the use of these analyses performed on different accessions of the species collected from geographically distinct areas of India. Our findings can be used for the commercial production and germplasm management of this medicinal plant. clusters with two samples as independent branches. As can be seen from the dendrogram, the grouping of samples did not follow any pattern related to their agro-ecological region or geographical distance. The dendrogram based on chromatogram was compared with that generated using the molecular technique ISSR and showed little correlation. Samples from Pratapgarh and Banasthali, Chittorgarh, and Roorkee showed similarity in chemotyping and ISSR. Despite some similarities, a distinct correlation between the grouping of samples based on chemoprofiling and molecular profiling could not be established. A study conducted in 2005 also could not correlate the genetic dendrogram with that based on essential oil content in Cymbopogon sp. [44]. This is understandable as chemoprofiling is influenced by the related climatic condition, geographic location, and

Conclusions
It has been well-documented that geographical conditions may affect the phytochemical profile of the plant. In addition to genetic research, phytochemical differences are investigated as a method for evaluating biodiversity and authenticating germplasm. This investigation revealed a large amount of variation across S. cumini accessions. The most effective accessions for improvement projects were identified using the ISSR marker system and the study of commercially significant phenolic acids. These accessions might be employed as acceptable feedstock for various commercial uses. Author Contributions: Conceptualization, S.K. and A.P.; methodology, S.K. and A.P.; software, S.A.; validation, S.K., K.S., and A.P.; formal analysis, A.P., S.K. and A.C.; investigation, S.K.; data curation, S.K.; writing-original draft preparation, S.A. and K.S.; writing-review and editing, S.K., A.P., and A.C.; visualization, S.K.; supervision, S.K. and A.P.; project administration, A.P. and A.C. All authors have read and agreed to the published version of the manuscript.